Cytotoxicity of CAR⁺PEBL T lymphocytes. (A) Four-hour cytotoxicity assays of PEBL or control (mCherry-transduced) T cells from 3 donors electroporated either with anti-CD19-41BB-CD3ζ CAR mRNA or no mRNA against CD19⁺ ALL cell lines at 2:1 E:T (see also supplemental Figure 4). Symbols indicate mean of 3 measurements for each donor. (B) Cytotoxicity of CAR-transduced T lymphocytes from 2 donors, sequentially transduced with a retroviral vector containing either mCherry alone or anti-CD3 PEBL was tested against CD19⁺ cell lines. Control, cells transduced with GFP only followed by mCherry only. Shown are data for 4-hour assays against CD19⁺ ALL cell lines at 1:1 E:T (full set of data in supplemental Figure 4). Each symbol indicates mean of triplicate experiments for each donor. (C-D) T lymphocytes transduced as in panel B were tested for long-term cytotoxicity against Nalm6 transduced with mCherry. Leukemia cell growth was measured with IncuCyte Zoom System (Essen BioScience). Whole-well imaging of triplicate cultures at 80 hours; E:T 1:8, is shown in panel C; leukemia cell growth measurements at the indicated E:T ratios in panel D. ***P < .001; ****P < .0001.